<p>Premature ovarian insufficiency (POI) is a common cause of female infertility, affecting approximately 1% of women under 40 years. The genetic etiology of POI exhibits significant heterogeneity, with a considerable number of cases remaining unexplained. In this study, we aimed to expand the genetic spectrum of POI by performing whole-exome sequencing (WES) in a cohort of 118 Chinese non-syndromic POI patients with secondary amenorrhea. A total of 115 possibly deleterious variants, 89 of which are novel, were identified in 58 known POI-causative genes, although some of these are classified as likely benign according to the American College of Medical Genetics and Genomics (ACMG) criteria. Specifically, 20 pathogenic/likely pathogenic variants were identified in 17 cases, seven of which were the likely genetic causes in these patients (7/118 cases, 5.9%). Another candidate gene list for POI, consisting of 93 genes, was compiled, drawing upon evidence from mouse models and functional studies, and 10 additional potentially deleterious variants were identified in 10 cases. Furthermore, a comparative analysis of POI cases across multiple studies showed that, while the genetic spectrum of POI varies among different populations, causative genes are predominantly involved in common biological processes including meiosis, DNA repair, gonad development, and germ cell development. In summary, using a cohort of 118 Chinese cases, we have broadened our understanding of the genetic spectrum of POI.</p>